Method in counterflow isotachophoresis

ABSTRACT

METHOD IN COUNTERFLOW ISOTACHOPHORESIS. INTO A COLUMN PROVIDED WITH TWO ELECTRODES A SAMPLE COMPRISING IONS OF DIFFERENT MOBILITY TO BE SEPARATED IS INTRODUCED TOGETHER WITH A FIRST ELECTROLYTE BETWEEN THE SAMPLE AND ONE OF THE ELECTRODES AND A SECOND ELECTROLYTE BETWEEN THE SAMPLE AND THE OTHER ELECTRODE. THESE FIRST AND SECOND ELECTROLYTES COMPRISE IONS OF HIGHER AND LOWER MOBILITY RESPECTIVELY THAN THE IONS OF SAID SAMPLE. A VOLTAGE IS APPLIED TO THE ELECTRODES AND A PRESSURE IS APPLIED TO ONE OF SAID ELECTROLYTES. A BOUNDARY BETWEEN ZONES OF IONS HAVING DIFFERENT MOBILITIES IS DETECTED AND FROM THIS DETECTION   A CONTROL SIGNAL IS GENERATED, SAID CONTROL SIGNAL CONTROLLING SAID PRESSURE AND/OR SAID VOLTAGE.

FIG. 1a

RIG 1' F. EVERAERTS METHOD IN COUNTERFLOW ISOTACHOPHORESIS Filed June 1,1971 DecQ 12, 1972 FIG. 2

United States Extent 3,705,845 METHOD IN COUNTERFLOW ISOTACHOPHORESISFrans Everaerts, Weert, Netherlands, assignor to LKB- Produkter AB,Bromma, Sweden Filed June 1, 1971, Ser. No. 148,657 Int. Cl. B01k 5/00US. Cl. 204-180 R 4 Claims ABSTRACT OF THE DISCLOSURE Method incounterflow isotachophoresis. Into a column provided with two electrodesa sample comprising ions of diiferent mobility to be separated isintroduced together with a first electrolyte between the sample and oneof the electrodes and a second electrolyte between the sample and theother electrode. These first and second electrolytes comprise ions ofhigher and lower mobility respectively than the ions of said sample. Avoltage is applied to the electrodes and a pressure is applied to one ofsaid electrolytes. A boundary between zones of ions having differentmobilities is detected and from this detection a control signal isgenerated, said control signal controlling said pressure and/or saidvoltage.

The present invention relates to a method in counterflowisotachophoresis, in which a sample, containing ions of the samepolarity which are to be separated, is introduced into a column arrangedbetween two electrodes, at which in that part of the column which issituated between the sample and that electrode towards which the ions inquestion are migrating when a voltage is applied to the electrodes, aleading electrolyte is introduced containing ions of still the samepolarity but with a higher mobility than that of the sample ions, andbetween the sample and the other electrode a terminating electrolyte isintroduced containing ions of still the same polarity but with a lowermobility than that of the sample ions, and that the above mentionedleading electrolyte is supplied to the column under a pressure so thatthe electrolyte is made flowing towards the sample.

Isotachophoresis (Greek: iso=equal, tacho=speed) is an electrophoreticseparatiofi method which is more closely described in e.g. Anal. Chim.Acta 38 (1967) 23 3-237, under the name of Displacement Electrophoresis.Isotachophoresis is principally carried out in that way that a samplecontaining a number of different ions with the same polarity isintroduced into some type of column in which the sample is locatedbetween two electrolytes, containing ions of the same polarity as thoseions in the sample which are going to be separated, whereat one of theelectrolytes contains ions with a higher mobility than that of thesample ions and the other electrolyte contains ions with a lowermobility than that of the sample ions. The electrolyte containing ionswith a higher mobility, the so called leading electrolyte, is at thatintroduced in that part of the column which is connected to theelectrode, towards which the sample ions are migrating, and theelectrolyte with the slower ions, the so called terminating electrolyte,is introduced at the other electrode. If a voltage is then applied tothe electrodes, the ions will be migrating in the column, at which theions of the sample will be separated according to their mobility. Thecourse thus obtained will be closer explained below.

The essential advantage of isotachophoresis in comparison withconventional electrophoresis is that substantially smaller amounts ofsample can be separated and that very sharp boundaries are achievedbetween the separated zones of sample, the zones of sample thus not be-Patented Dec. 12, 1972 ing extended during the course of separation asin conventional electrophoresis. A disadvantage is, however, that a longcolumn is to be used in working with very small ions concentrations andin working with sample ions with very slight diiference in mobility,whereby very high potentials are required to give sufficient fieldstrength in the column. The length of the column might, however, beconsiderably reduced if a counterflow of the leading electrolyte isutilized, i.e. the leading electrolyte is pumped against the migrationdirection of the sample ions. (See e.g. Preetz and Preifer, Anal. Chim.Acta 38 (1967 255- 260.) In that case a separation can be achievedwithout moving in the column the boundary between the sample and theleading electrolyte. By shortening the column the requisite fieldstrength can be obtained by considerably lower potentials. The problemis, however, to choose the counterfiow and the electric current in thecolumn in such a way that one of the zones, which are obtained when thesample is separated, is mainly immobile until the separation iscompleted and until an equivalence state has ensued. To solve thisproblem is hitherto proceeded in that way that the boundary between theleading electrolyte and the first zone of the sample is observated andthe counterflow is being adjusted e.g. by means of a verticallyadjustable reservoir, connected to the column and containing the leadingelectrolyte, as soon as the boundary has any tendency to move in eitherdirection. This, however, brings about that the device must be keptunder observation during all the separation course and it is furtherdifiicult manually to accomplish the exceedingly small variations of thecounterfiow which are necessary in order that the separation should notbe disturbed. Thus the purpose of the present invention is to provide amethod in counterflow isotachophoresis in which those disadvantages areeliminated.

The invention will now be closer explained with reference to theaccompanying drawing in which FIGS. la and 1b schematically show how thedifferent ions of a sample are separated, and

FIG. 2, also schematically, shows a device for accomplishing the methodaccording to the invention.

In FIGS. 1a and lb the number 1 denotes a column in which an anode 5 anda cathode 4 are introduced. It is further assumed that the sample whichis to be separated is introduced into that part of the column which isdesignated with S, and that the sample consists of salts containing twodiiferent anions C and C of which C is assumed to have a higher mobilitythan C and a common cation R+. The part of the column which isdesignated L is filled with the above mentioned leading electrolyte,which consists of anions A with a higher mobility than all the anions ofthe sample and a cation, designated R+, suitably common with the sample.The part T of the column which is turned towards the cathode is filledwith an electrolyte which contains an anion B-, the mobility of which islower than of all anions of the sample, and a cation R+, common with theleading electrolyte. When a direct current voltage is applied to theelectrodes 4 and 5 the cations will migrate towards the cathode 4 andthe anions will migrate towards the anode 5. As a result of thedifferent mobilities of the anions a zonewise stepwise increasingpotential gradient will then be obtained over the zones L, S, and Trespectively. The potential gradient over the zone S will, however, leadto that the anions in the zone will be separated according to theirmobility, so that those ions C," with the higher mobility will locate.themselves nearest to the leading electrolyte, and those ions C havingthe lower mobility will locate themselves nearest to the zone T.Provided that a mainly constant current is forced through the column,the anions of the sample will thus be separated and after the separationthe ditferent zones in the column will migrate stable since an anionwhich e.g. is ditfusing into an ahead lying zone with a lower potentialgradient will get a lower speed so that it will be brought back to itsoriginal zone. In the same way an anion which is diflusing into a rearzone because of the potential gradient present in that zone will bebrought back into its original zone. A very good self-stabilizing of theZone boundaries is thus achieved. In order to detect the different zonesand their lengths the stepwise increasing potential gradient is suitablyutilized. For example the correspondingly stepwise increasingtemperature on the outer surface of the column 5 could be measured orthe potential at one or more points on the column could be directlymeasured. Of course it is also possible, by means of the electrode, tomeasure the conductivity of the zones passing a certain point, and sofar the separated substances are UV-absorbing, the UV- absorption couldalso be measured in conventional manner.

As has been mentioned in the introduction it is, however, a disadvantageof the separation method described above, that a rather long column isrequired for the separation, especially when the difference is smallbetween the mobilities of the different ions in the sample, why in orderto achieve the requisite field strengths a very high voltage must beapplied to the electrodes with accompanying problems of construction andof safety. The

length of the column can, however, be considerably reduced, if leadingelectrolyte is continuously pumped to the column during the separation.The magnitude of the counterflow can then suitably be chosen in a waythat the boundary between the zones L and S principally is tainingleading electrolyte has been raised or lowered.

This procedure has, however, the drawback that the sample must be keptunder permanent observation and further an incautious action on theliquid level might bring about that the magnitude of the counterflow isaltered in such a way that the separation is seriously 4.5

disturbed. The purpose of the present invention is therefore to providea procedure by means of which the counterflow always is given such avalue that a certain zone boundary is occupying a fixed position. Theinvention will now be more closely explained with reference to FIG.

2, which schematically shows a device for performing the procedureaccording to the invention.

In FIG. 2 the number 1 denotes a column, which is arranged between ananode chamber 3 and a cathode chamber 2. In-the electrode chambers ananode 5 and a cathode 4 are respectively introduced and these areconnected to a current source 6. The column is further equipped with amultiway valve 7 by means of which the column could either be connectedto the chamber 2 or to a sample injecting means 8. By this injectingmeans 30 the sample can be introduced into a capillary between theterminating electrolyte, which is introduced into the cathode chamber 2,and the leading electrolyte, which is introduced into the anode chamber3 and in addition into that part of the column which is situated betweenthe 5 valve and this chamber. That part of the column which is situatedat the anode is further suitably equipped with a valve 10 via whichleading electrolyte could be supplied or removed. Further, a diaphragm20 is arranged between the column and the anode chamber 5 in a way thatthe column could be flushed with liquid without changing thecircumstances in the chamber. With those polarities of the electrodes 4and 5 which have been chosen in the figure, it is then assumed that theanions of the sample are the subjects of separation. If the sample onthe other hand would contain a number of diflerent cations the reversepolarity of the electrodes would of course be chosen. The device isfurther equipped with means for varying the counterflow of the leadingelectrolyte. This means consist of a reservoir 11, in which a floater 13can be moved up and down so that the liquid level in the reservoir andthus the pressure of the counterflow can be varied. A spindle 14 whichis supporting the floater is introduced into an electromagnet 15 so thatby varying the current in the electromagnet the floater is brought upand down in the reservoir. The governing of the current through theelectromagnet is accomplished by means of a detection means 17 connectedto the column. The detection organ can be thermocouple but can alsoconsist of any other of the detecting elements which has been statedabove in connection to FIGS. la and lb. The thermocouple is connected toan amplifier 18, the output of which in its turn is connected to agoverning device 19. This one is in its turn connected to theelectromagnet 15. The signal from the thermocouple should then governthe counterflow in order that a zone boundary, suitably the boundarybetween'the leading electrolyte and the first sample zone, is mainlystationary at the thermocouple. As hinted above there will be obtained atemperature rise at this boundary which temperature rise also will causea temperature rise on the outer surface of the column. Thus thegoverning device 19 should principally give an output signal which keepsthe floater 13 in a fixed position when the signal from the thermocouplecorresponds to a temperature which constitutes the mean value betweenthe temperature in the leading electrolyte and that in the first samplezone. A signal corresponding to a lower temperature should further causea slow decreasing of the coun terflow by means of raising the floater13, and a signal corresponding to a higher temperature shouldcorrespondingly cause a slow increase of the counterflow, i.e. alowering of the floater 13. In this connection it should be stated thatcounterflow governing by means of a vertically adjustable floater ofcourse only is one example of varying the eounterflow. A great advantageof the shown counterflow governing is, however, that very smallcounterflow alterations, free from pulsation, can be achieved, to a veryinconsiderable extent disturbing the separation in the column. However,any pressure generating means could be generally utilized. As appearsfrom the drawing the governing device 19 is also connected to a currentsource 6, and the purpose of this connection is that by varying theelectric current in the column in dependence of the current from thethermocouple a certain zone boundary should be kept in a fixed position.Of course it is also possible to fix the zone boundary by alfecting boththe counterflow and the electric current.

We claim: 1. Method in counterfiow isotachophoresis comprising the stepsof:

introducing into a column provided with first and second electrodes asample comprising ions of the same polarity to be separated, a firstelectrolyte between the sample and said first electrode and a secondelectrolyte between the sample and said second electrode, said first andsecond electrolytes comprising ions of higher and lower mobilitiesrespectively than the ions of said sample; applying a voltage betweensaid first and second elec trodes, the voltage having such a polaritywhich will give the sample ions a tendency to migrate towards said firstelectrode; applying a pressure difference between said first and secondelectrolytes; detecting by a detection means a boundary between zones ofions having different mobilities; and obtaining a control signal fromsaid detection means, the. control signal controlling said pressure inorder to govern said boundary to move with a desired velocity.

2. Method as recited in claim 1, wherein said velocity is zero.

3. Method in counterflow isotachophoresis comprising the steps of:

introducing into a column provided with first and second electrodes asample comprising ions of the same polarity to be separated, a firstelectrolyte between the sample and said first electrode and a secondelectrolyte between the sample and said second electrode, said first andsecond electrolytes comprising ions of higher and lower mobilitiesrespectively than the ions of said sample;

applying a voltage between said first and second electrodes, the voltagehaving such a polarity which will give the sample ions a tendency tomigrate towards said first electrode;

applying a pressure difi'erence between said first and secondelectrolytes;

detecting by a detection means a boundary between zones of ions havingdifferent mobilities; and

UNITED STATES PATENTS 2,711,379 6/1955 Rothstein 204180 R X 3,305,471 2/1967 Von Miinchhausen et a1.

204-180 R X 3,384,564 5/1968 Ornstein et a1. 204-180 G 3,533,933 10/1970Strauch 204-180 G JOHN H. MACK, Primary Examiner A. C. PRESCOTT,Assistant Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE, CERTIFICATE OF CORRECTION Patent No. 3,705,5 Dated m r 1 19-72 Inventods) Frans Everaerts It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

In the heading, after "Filed June 1, 1971, Ser'. No.

l I8,657" add Claims Priority Swedish Application No.

762 I/7O, filed June 2, l970- Signed and sealed this 8th day of May1973.

(5L aL) Attest:

ED 5431) 1-5.. FLETGHER,J'R. ROBERT GOTTS'CHALK attesting OfficerCommissioner of Patents FORM PO-IOSO (IO-69) USCOMM-DC 60375-p59 U45,GOV ERNMENT PRINTING OFFICE: I969 O366'334 I

